Myotonic dystrophy type 1 (DM1) differs from other forms of muscular dystrophy in having an unconventional mutation and a novel disease mechanism. The genetic lesion is an expanded CTG repeat in DMPK, and the mechanism involves a toxic gain-of-function by transcripts containing an expanded CUG repeat (CUGexp). Recent studies have suggested that these unique characteristics create a major therapeutic opportunity. In rapid succession, therapeutic targets responsible for RNA toxicity were identified, agents acting on targets were developed, and reversal of the disease, at least in its early stages, was accomplished in mouse models. As new putative therapies advance to clinical trials, there is a need to obtain conclusive evidence for or against target engagement, at the earliest possible moment, using biomarkers of therapeutic response. The goal of this proposal is to develop assays and biomarkers for this purpose. Major emphasis is placed on alternative splicing because previous work has suggested that splicing misregulation is the best overall readout for downstream effects of RNA toxicity. In addition, analytical methods to assess splicing regulation are very precise, and studies of targeted therapy in mouse models have shown that splicing defects are fully reversible and closely aligned with functional improvement. Building on these observations, and a comprehensive ascertainment of DM1-associated splicing defects from several large data sets, we propose in Aim 1 to develop Multiplex Alternative Splice sequencing (MASseq), a targeted high-throughput sequencing method to assess splicing biomarkers in small muscle biopsy samples. In an effort to qualify MASseq for biomarker analysis, we will optimize the method and then systematically examine sampling variance, assay variance within and between runs, and test-retest reliability. The latter studies will use paired muscle samples from the same subjects, collected eight weeks apart. To test fidelity and assess bias, MASseq results will be compared to reference data from conventional RNAseq of the same samples. Finally, associations of MASseq with functional impairment will be examined. While splicing biomarkers are applicable across all therapeutic strategies for DM1, our second Aim is specifically designed for treatments that reduce cellular levels of toxic RNA. We will develop methods to quantify allelic expression of DMPK, by determining the ratio of mutant versus wild-type transcripts. We will also optimize a novel method to measure the total cellular burden of CUGexp RNA. The results of this project will provide useful biomarkers to guide decision making in clinical trials and potentially in clinical practice, using methods that ae readily transferable to other laboratories.